In year 3 we will continue the characterization of the two types of N2ase Fe protein found in photosynthetic bacteria. The enzyme from glutamate-or N2-grown cells is inactive in vitro due to a ribose-phosphate-adenine moiety covalently bound to the Fe protein; Fe protein from N-starved cells is unmodified and fully active. It will be determined if the in vitro activated Fe protein is identical to that from N-starved cells or constitutes a second type of active Fe protein. The activation by the Mn2 ion-dependent activating enzyme was previously reported by others to cleave only the adenine unit from this group. Activation of 32P-labeled Fe protein has shown that phosphate is also cleaved from the Fe protein. We now plan to grow R. rubrum in media supplemented with 14C-ribose, first to determine if 14C-Fe protein can be obtained, and if it can be, to determine if the ribose portion of this modifying group is removed during activation. If it is, it would indicate that active N2ase from N-starved cells is identical to that which results from in vitro activation. A second line of research to be pursued relates to the enzyme system in R. rubrum that inactivates the N2ase Fe protein. Preliminary indications suggest glutamine synthetase may be involved in this inactivation process. We intend to isolate glutamine synthetase and glutamate synthase negative mutants and then examine the N2ase regulatory patterns in these auxotrophs. If one of these enzymes is involved in the regulation of Fe protein, the regulatory pattern should vary in one of these mutants. Finally, we intend to purify to homogenity the Fe protein activating enzyme from R. rubrum and determine its subunit composition and metal requirements for turnover. While we have already characterized this enzyme in a preliminary way, only analytical quantities have been purified and these have not been sufficient for a thorough chemical and enzymatic analysis which is intended for the pure enzyme.